Staphylococcal enterotoxins (Ents) are part of a larger family of pyrogenic toxins (PTs) produced by Staphylococcus aureus and Streptococcus pyogenes. Although they are known mainly for their ability to cause food poisoning, Ents, like other PTs, can cause toxic- shock syndrome (TSS) as the result of a set of shared immunomodulating biological activities. Sequence similarities among PTs suggests that the toxins are related at the molecular level. This project will explore the hypothesis that shared PT properties are the result of conserved protein structure and that it may be possible to map conserved functional regions on the toxins. Type C Ents will serve as representative models for the PT family since they cause both TSS and food poisoning. Molecular regions and amino acids required for toxicities linked to both illnesses will be identified (Aims 1 and 2). Genetically-truncated Ent molecules, chemically-fragmented Ents, and synthetic peptides will be tested for emetic activity in cats and monkeys, and TSS-associated properties in several in vitro and in vivo assays. Important amino acids will be identified by site-directed mutagenesis. To elucidate the mechanism by which PTs cause TSS, altered Ents, deficient in a biological activity, will be tested in a rabbit model (Aim 3). Also, the virulence of S. aureus strains, differing only by modification of their ent genes, will be compared in rabbits. To further evaluate the relatedness of PTs, specific and shared antigenic epitopes will be mapped on type C Ents (Aim 4). Specific and cross- reactive antibodies (monoclonal and polyclonal) will be used to map epitopes by their reactivity with toxin fragments and peptides. Finally, Ent C1 crystals will be produced for X-ray diffraction analysis to determine toxin 3-dimensional structure (Aim 5). This detailed analysis of the structural and functional organization of Type C Ents will provide information regarding the PT family, their molecular and possible evolutionary relatedness, and the mechanisms by which they cause human disease. Because of the similarities that type C Ents have with other PTs, this information may be useful for designing nontoxic vaccines against several staphylococcal and streptococcal toxic diseases.